Antibodies are molecules that provide a key defense against infection in humans. They are used as therapeutics in treatment of a variety of diseases, from infectious disease to cancer. They are also used as diagnostic reagents in a huge variety of tests carried out daily in clinical and research laboratories.
Antibody specificity and affinity are modified in vivo by processes of mutation, targeted to specific regions within the genes that encode antibodies. Antibodies are encoded by two genes, referred to as the immunoglobulin (Ig) heavy and light chain genes. The heavy and light chains of polypeptides encoded by the Ig genes interact to form a tetrameric molecule which is expressed on the cell surface as a receptor. Antibody molecules are bifunctional: one domain recognizes antigen, the other promotes removal of antigen from the body. The recognition domain, referred to as the variable (V) region, is created upon interaction of the heavy and light chain polypeptides in natural antibodies. It is in fact variable in sequence from one antibody to another. Variability in V region primary sequence (and hence three-dimensional structure and antigen specificity) is the result of processes which alter V region sequence by causing irreversible genetic changes. These changes are programmed during B cell development, and can also be induced in the body in response to environmental signals that activate B cells. Several genetic mechanisms contribute to this variability. Two subpathways of the same mechanism lead to two different mutagenic outcomes, referred to as somatic hypermutation and gene conversion (reviewed (Maizels, 2005)). Somatic hypermutation inserts point mutations. Somatic hypermutation provides the advantage of enabling essentially any mutation to be produced, so a collection of mutated V regions has essentially sampled a large variety of possible mutations. Gene conversion inserts mutations that are “templated” by related but non-identical sequences. Gene conversion provides the advantage of creating a repertoire based on information already stored in the genome, which may be optimized by evolutionary selection.
The modified antigen receptor is the target for selection. Ig is expressed on the cell surface, which permits clonal selection of B cells with desired specificity or affinity in a physiological context or within cultured cells. Cell surface Ig can readily be detected by flow cytometry of cells stained with anti-Ig. Binding of Ig molecules to specific compounds can be detected as interaction with fluorescent derivatives of those compounds, analyzed by flow cytometry; and B cells that bind to specific compounds can also be recovered upon sorting by flow cytometry. B cells that bind to specific compounds can also be selected on solid supports carrying those compounds. Conversely, binding to solid support also permits removal of B cells with unwanted binding specificities. Repetitive cycles of binding and release permit enrichment for high affinity binding.
Mutation and gene targeting occur in the DT40 B cell line. DT40 is a chicken B cell line that proliferates readily in culture. DT40 has been widely documented to carry out constitutive mutagenesis of its heavy and light chain immunoglobulin genes (Reynaud et al., 1987; Thompson and Neiman, 1987; Reynaud et al., 1989). Mutagenesis is targeted to the V regions. Mutations are normally templated by copying related non-functional V gene segments, called “pseudo-V” genes, which are present in a linear array upstream of each functional V region. Templating is evident as tracts of sequence shared between the mutated V target and one of the pseudo-V genes. Genetic alterations of DT40 have been shown to cause a switch from templated mutagenesis to nontemplated mutagenesis, which results in somatic hypermutation, by a pathway essentially identical to somatic hypermutation that alters the sequences of V regions in human B cells (Sale et al., 2001). DT40 has also been widely documented to support very efficient homologous recombination, or gene targeting (Buerstedde et al., 2002; Sale, 2004). This enables creation of derivatives in which specific genes or genomic regions have been modified or ablated; or in which one genetic region has been replaced with another.
Due to the limitations and challenges posed by currently available approaches to targeted mutagenesis there is a need in the art for the development of alternative methods and constructs. The present invention fulfills this need, and further provides other related advantages.